Transglutaminases are calcium-dependent enzymes which catalyze acyl transfer reactions that lead to the amination of proteins with small primary amines or the crosslinking of proteins by the formation of .epsilon.(.gamma.-glutamyl)lysine transpeptide bonds. This crosslinking activity typically results in the covalent joining of soluble proteins into large chemically, enzymatically and mechanically resistant polymers with diverse biological functions.
Several classes of transglutaminase exist and form a family of genes. The "a" subunit of factor XIII (XIIIa) participates in blood clot formation by crosslinking fibrin. Factor XIIIa is a soluble protein in the plasma and in placenta; it is also know as plasma TGase. TGase I is a differentiation marker for keratinocytes and plays a role in formation of the cornifying envelope in epidermal keratinocytes. TGase I is predominantly membrane-bound, and has been variously identified in the literature as particulate TGase, epidermal TGase, and TGase K. TGase II is ubiquitously distributed in the cytosol. The physiological role of TGase II is not well understood. This enzyme is also known as tissue TGase, liver TGase, intracellular TGase, soluble TGase and TGase C. Band 4.2 protein also belongs to the TGase gene family although this protein lacks transglutaminase activity.
TGase I gene expression appears to be limited to the terminally differentiating keratinocyte in vivo and in vitro. In these cells, the formation of an insoluble protective structure, the cell envelope, just beneath the plasma membrane is associated with increased crosslinking activity of TGase, which in turn is correlated with an increased level of TGase I mRNA [Polakowska et al. (1991) J. Invest. Dermat. 96:285-288]. TGase I expression, as well as terminal differentiation, are regulated by several environmental factors of which Ca.sup.2+ and retinoic acid (RA) appear to have the most profound effect. Hence, TGase I provides a convenient marker for keratinocyte differentiation.
A cDNA clone for the human TGase I gene, and the sequence thereof has been reported by several investigators [Kim et al. (1991) J. Biol. Chem. 266:536; Phillips et al. (1990) Proc. Natl. Acad. Sci. USA 87:9333; Polakowska et al. (1990) J. Invest. Dermat. 94:567; Polakowska et al. (1991) J. Invest. Dermat. 96:285; Schroeder et al. (1990) Clin. Res. 38:962A; Yamanishi et al. (1991) Biochem. Biophys. Res. Comm. 175:906]. However, the genomic gene for human TGase I with its concomitant regulatory regions has heretofore been unavailable and therefore uncharacterized.
The present invention arose from the identification and characterization of the human TGase I genomic DNA. Accordingly, once the human genomic DNA was available, the regulatory regions, or elements, responsible for initiating and controlling transcription of human TGase I were elucidated as provided by the present invention. Since TGase I is expressed in a tissue-specific manner, i.e. in terminally differentiating keratinocytes, the subject regulatory regions are particularly useful in construction of tissue-specific expression vectors as well as to provide promoter elements responsive to calcium and retinoic acid. Moreover, each of the regulatory elements of the present invention is useful in combination with known promoter or regulatory elements. These combinations enable modification of the host-cell or condition responsiveness of other vectors and thus provide additional means to control gene expression. Such combinations provide greater flexibility in selecting vectors to control expression in a selected cell type or under a particular set of conditions.
By identifying and characterizing the human TGase I promoter elements and linking these elements to marker genes, other factors which may regulate expression, such as pharmaceutical and cosmetic agents for the skin, can be examined. Further, using the promoter regulatory elements identified by the present invention, expression vectors can be constructed which direct expression of heterologous gene products in skin equivalents or which direct altered expression of TGase in skin equivalents. Skin equivalents, or artificial skin, are epidermal cells grown in tissue culture which have many uses including skin grafts, especially for burn victims, and testing pharmaceutical and cosmetic products as well as the individual constituents of such products.
Hence, the present invention provides expression vectors containing the transcriptional regulatory region(s), (i.e. elements of the promoter region) of the human TGase I gene which are particularly useful for human gene therapy to treat diseases of the epidermis or to deliver drugs in a cell-specific manner, for production of skin equivalents, and for the use with skin equivalents to provide in vitro testing of pharmaceutical and cosmetic products.